The long term goal of this research project is to understand the molecular mechanism of force production through 3-D visualization of myosin molecular motors in situ in muscle. The research focuses on the structure of the large waterbug Lethocerus sp. because its filament lattice is the best ordered of all known muscles types thereby making it an excellent candidate for 3-D imaging as well as facilitating the trapping of many myosin motors into similar states. Lethocerus, like many insects, utilize a stretch activation mechanism to operate their flight muscles. Stretch activation also occurs in vertebrate striated and cardiac muscle, where in the case of cardiac muscle, it is an important part of the rhythmic contractions. Specimen preparation emphasizes rapid freezing and freeze substitution which traps molecular motions with millisecond time resolution. The structure of isolated Lethocerus thick filament in the relaxed state will be investigated using electron cryomicroscopy. Myosin motors during isometric contraction itself and following mechanical perturbations such as quick stretch and release will be trapped by fast freezing and imaged in 3-D with the specific aim of obtaining a higher resolution structure. Rigor fibers swollen in low ionic strength buffers enhance the visibility of the 1-helical coiled-coil domain that links the myosin head to the thick filament backbone. The specific aim of this study will be to derive structural rules that define the position on the thick filament from which force producing myosin heads must originate and thereby test a new model for the weak-to-strong binding transi- tion in myosin. Specimens with enhanced numbers of weak binding myosin heads will be trapped by rapid freezing to further improve their characterization. Particular emphasis will be place on myosin heads binding to troponin, which may be important players in the stretch activation mechanism of muscle contraction. Improvements to increase structure homogeneity are pro- posed so that higher resolution images of active molecular motors can be obtained to better de- fine structural intermediates in the weak-to-strong transition and in force production itself. We will utilize electron tomography to obtain 3-D images of individual muscle motors and use multivariate data analysis to identify groups having similar structure for subsequent averaging to improve the signal-to-noise ratio and resolution. Continued refinements of our unique tomographic methods are proposed in order to increase resolution, improve the reconstructions and the rate at which 3-D maps can be produced. Atomic models based on the crystal structures of actin and myosin will be quantitatively fit within the envelope of the reconstruction and used to make pre- dictions of domain orientations, actin binding affinity and progress through the working stroke. PUBLIC HEALTH RELEVANCE: One of the characteristics of cardiac muscle is its response to a stretch, which generally causes a delayed increase in tension. The molecular basis of stretch activation is unknown. Stretch activation is critical to flight by certain genera of insects making them an excellent model system with which to investigate this phenomenon.